Fragments of a lymphocyte adhesion receptor for high endothelium, CD44

ABSTRACT

A lymphoid cell line cDNA that encodes an adhesion receptor for high endothelial venules (HEV).

This invention was made in part with government support under GrantCA15704 and National Cancer Institute grants CA42571 and R01 CA40272.The government has certain rights in this invention.

This application is a divisional of Ser. No. 07/884,624, filed May 15,1992 (U.S. Pat. No. 5,504,194), which is a continuation of Ser. No.07/628,646, filed Dec. 12, 1990 (now abandoned), which is a divisionalof Ser. No. 07/325,224, filed Mar. 17, 1989 (U.S. Pat. No. 5,002,873).

TECHNICAL FIELD

This invention relates to genetic engineering involving recombinant DNAtechnology, and particularly to the identification of a DNA sequenceencoding a lymphocyte adhesion receptor for high endothelium.

BACKGROUND OF THE INVENTION

Circulating lymphocytes traffic among the blood vasculature, thelymphatic system, and sites of chronic inflammation facilitatinginteractions among lymphocytes, antigens, and accessory cells thatultimately lead to the generation and dissemination of an immuneresponse. Entry of blood-borne lymphocytes into the lymphoid organsentails adhesion to the postcapillary endothelia followed byextravasation. For reviews, see: Jalkanen, S. T., et al., Immunol. Rev.91: 39-60, 1986a; Woodruff, J. J., and L. M. Clarke, Ann. Rev. Immunol.5: 201-222, 1987; Gallatin, M., et al., Cell 44: 673-680, 1986. Inperipheral lymph nodes, mucosal lymphoid organs (Peyer's patches andappendix), and inflamed synovia, this adhesion-mediated entry occursprimarily at specialized high-walled endothelial cells lining thepostcapillary venules (HEV). Adhesion and possibly transmigration acrossthe venule is postulated to be mediated by a specific lymphocyte surfacereceptor or receptors interacting with complementary HEV molecules.These lymphocyte adhesion receptors (also referred to as homingreceptors) have been implicated in the interaction of other nonlymphoidhematopoietic cells with vascular endothelia (Lewinsohn, D. M., et al.,J. Immunol. 138: 4313-4321, 1987), and are postulated to play a role inthe metastasis of lymphoid tumors (Jalkanen, S. T., et al., 1986a,supra). There is mounting evidence, discussed below, that these adhesionreceptors may function widely in other tissue systems as well.

Studies conducted both in vivo and in vitro revealed that subsets oflymphocytes preferentially migrate to or adhere to the HEV of differentlymphoid organs, suggesting the involvement of multiple adhesionreceptors with different specificities. Griscelli, C., et al., J. Exp.Med. 130: 1427-1451, 1969; Guy-Grand, D., et al., Eur. J. Immunol. 4:435-443, 1974; Scollay, R., et al., Nature 260: 528-529, 1976; Smith, M.E., et al., Monogr. Allergy 16: 203-232, 1980; Butcher, E. C., et al.,Eur. J. Immunol. 10: 556-561, 1980; Stevens, S. K., et al., J. Immunol.128: 844-851, 1982. Some lymphoid tumors were found to express aunispecific preference for the HEV of either peripheral lymph node orfor gut-associated lymphoid tissue (Butcher, E. C., et al., 1980,supra). These data suggest the participation of at least two distinctadhesion receptors that confer lymphoid organ specificity, contributingto the migratory patterns of lymphocytes.

Direct evidence for the presence of multiple adhesion receptors emergedin rodents, humans, and nonhuman primates as immunological reagentsrecognizing these molecules became available. MEL-14 is a monoclonalantibody raised against a 90 kD cell-surface protein present on aperipheral node HEV-binding mouse lymphoma, 38-C13 (Gallatin, W. M., etal., Nature 304: 30-34, 1983). The mouse protein defined by MEL-14 isglycosylated and ubiquitinated, and contains internal disulfide bonds(Siegelman, M., et al., Science 231 :823-829, 1986; St. John, T., etal., Science 231: 845-850, 1986). MEL-14 reactivity correlates withperipheral node HEV-binding specificity of B and T cell tumors; and wheneither normal lymphocytes or unispecific tumor cells are pretreated withthe MEL-14 antibody, in vitro adhesion to peripheral node but notPeyer's patch HEV is blocked, and migration in vivo to peripheral nodesis selectively diminished (Gallatin, W. M., et al., 1983, supra).

Monoclonal antibodies (mabs) that identify glycoproteins with similarfunctions in primates have also been characterized: Hermes-1, whichrecognizes migratory competent and HEV-adherent human lymphocytes(Jalkanen, S. T., et al., Eur. J. Immunol. 16: 1195-1202, 1986b);Hermes-3, which specifically blocks lymphocyte binding to human appendixand Peyer's patch HEV (Jalkanen, S. T., et al., J. Cell Biol. 105:983-990, 1987); and Hutch-1, which defines related molecules in macaques(W. M. Gallatin, unpublished data). All of the epitopes recognized bythese mabs reside on the same molecule. Although the MEL-14 and Hermes-1receptors are apparently immunologically related (Jalkanen, S. T., etal., 1987, supra; Jalkanen, S., et al., J. Immunol. 141: 1615-1623,1988), no direct evidence indicates that they are the products ofhomologous genes.

Biochemical similarities between the Hermes/Hutch class of adhesionreceptors and the class III extracellular matrix receptor (ECMRIII), amolecule postulated to function as a transmembrane link between theextracellular matrix and the cytoskeleton (Carter, W. G., and E. A.Wayner, J. Biol. Chem. 263: 4193-4201, 1988), led to a detailedcomparison of the two receptors (unpublished data; T. P. St. John, W. M.Gallatin, et al.). These studies identified extensive structuralhomology between these molecules. The tissue distribution of ECMRIII isquite broad, including granulocytes, monocytes, fibroblasts, severalepithelial carcinomas, as well as lymphocytes, indicating that thesereceptors may serve an adhesive function in other tissue systems.

Other classes of adhesion receptors also contribute to lymphocyte-HEVinteraction. The integrin LFA-1 is important in the homotypic adhesionof activated lymphocytes and in the adhesion of T lymphocytes toendothelium (Haskard, D., et al., J. Immunol. 137: 2901-2906, 1986).Antibodies to LFA-1 partially inhibit lymphocyte adhesion to peripherallymph nodes in vitro and in vivo (Hamann, D. A., et al., J. Immunol.140: 693-699, 1988). However, in cell lines expressing high levels ofthe MEL-14 antigen, anti-LFA-1 treatment resulted in only minorreductions of HEV adhesion levels in contrast to treatment with MEL-14antibody, which essentially eliminated adhesion to HEV. These resultssuggest that LFA-1 may play an accessory role in some lymphocyte-HEVinteractions.

The study of HEV adhesion in the mouse has been facilitated by theexquisitely specific mab MEL-14. In the human and primate systems, themab recognizing apparently related molecules often fail to blockadhesion. The mechanism of organ specific HEV adhesion is unknown, as isthe molecular basis for the functional diversity among these molecules.

SUMMARY OF THE INVENTION

We report here the molecular cloning from a baboon lymphoid cell line ofa cDNA that encodes an adhesion receptor for HEV. The 362 amino acidprotein encoded by this cDNA is unique and not present in all thedatabases examined. The mature protein, resulting from the cleavage of aputative 20 amino acid signal peptide, has a calculated molecular weightof only 37 kD, indicating that the 90 kD cell surface protein is highlymodified. The 342 amino acids, which lack any repeated sequences ofsignificant length, encompass an extracellular domain (250 amino acids),a putative transmembrane domain (20 amino acids), and a cytoplasmicdomain (72 amino acids). After the cDNA sequences have been incorporatedinto replicable expression vectors, and the vectors transfected into anappropriate host (e.g., a mammalian, bacterial, or insect cell culture),the expressed polypeptide or polypeptides can be used to modulatemammalian immune function in at least two ways. In a firstrepresentative embodiment, the expressed product is administered in vivoin order to bind to and competitively block the lymphocyte adhesionsites on high-walled endothelial cells of postcapillary venules (HEV),and thereby prevent lymphocyte adhesion and extravasation at thecomplementary sites. In a second representative embodiment, theexpressed product is employed as an immunogen in order to raiseantibodies against lymphoid receptors for this specialized venuleendothelium. The antibodies are in turn administered to directly blocklymphocyte adhesion to HEV sites and thereby prevent or modulate themigration of lymphocytes from the bloodstream into the secondarylymphoid organs. Such antibodies are also useful for identifyingheretofore unknown cell populations, e.g., bone marrow subsets ofinterest.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 indicates the alignment of isolated cDNA clones encoding thelymphocyte adhesion receptor/ECMR III molecule, and structural featuresof the protein encoded by the prototype B7 cDNA clone, as described inExample 1;

FIG. 2 is an autoradiogram showing antibody binding to WGA-purifiedproteins from a 594S cell lysate by the crude R1594 antiserum and byantibody subsets affinity selected on the fusion protein products of theindicated individual cDNA clones, as discussed in Example 2;

FIG. 3 presents autoradiograms of the purified adhesion receptor protein(A), with (+) and without (-) subsequent cleavage by cyanogen bromide(B), as described in Example 3; and

FIG. 4 shows the DNA sequence and the translated protein sequence of theprototype B7 adhesion receptor cDNA clone, as described in Example 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As noted above, the migration of lymphocytes from the bloodstream intothe secondary lymphoid organs, necessary for a successful immuneresponse, occurs primarily within postcapillary venules that arecharacterized by high-walled endothelial cells. Lymphocyte adhesion toand extravasation at these sites is associated with the expression ofspecific lymphoid receptors for this specialized venule endothelium. Wereport here the molecular cloning from a baboon lymphoid cell line of acDNA that encodes an adhesion receptor for HEV. The cDNA clone wasidentified by antibody screening of cDNA-encoded fusion proteinsfollowed by epitope selection analysis. This analysis indicated that atleast two independent epitopes are detected by the antiserum probe.Amino acid sequence data on purified protein independently confirmed itsidentity. The 362 amino acid protein encoded by this cDNA is unique andnot present in all the databases examined. The mature protein, resultingfrom the cleavage of a putative 20 amino acid signal peptide, has acalculated molecular weight of only 37 kD, indicating that the 90 kDcell surface protein is highly modified. The 342 amino acids, which lackany repeated sequences of significant length, encompass an extracellulardomain (250 amino acids), a putative transmembrane domain (20 aminoacids), and a cytoplasmic domain (72 amino acids).

It is contemplated that soluble polypeptides corresponding to theextracellular domain (amino acids 21-270) shown in FIG. 1, or antibodiesdirected thereto, can be administered in vivo to block, by competitiveinhibition or direct interference, the entry of circulating lymphocytesinto peripheral lymph nodes, mucosal lymphold organs, and/or inflamedsynovia. By modulating such adhesion-mediated entry in these ways, thetissue-specific effects of the lymphocytes can be regulated fortherapeutic purposes. For example, the engraftment of allogeneic tissuesuch as bone marrow can be enhanced in this manner. Autoimmune diseasestates such as rheumatoid arthritis can also be treated in this manner.Other examples include treatment of unreactive colitis, encephalitis,and other chronic site-specific inflammatory conditions.

For such purposes, the soluble external domain will often be employed,typically but not necessarily polymerized in a multivalent state using,e.g., dextran or polyamino acid carriers. Liposomes may alternatively beemployed as the therapeutic vehicle, in which case the transmembranedomain (amino acids 271-290 in FIG. 1) and preferably at least some ofthe cytoplasmic domain (amino acids 291-362 in FIG. 1) will also beincluded.

For treating certain clinical conditions, it may be advisable to removeendogenous soluble receptor from a patient's blood serum, and this canbe done with existing on-line and off-line techniques by employingimmunoselection columns containing antibodies directed against thedisclosed external domain.

The external domain can also be employed for targeting therapeutic anddiagnostic moieties to HEV binding sites. For example, toxin- orradionuclide-bearing conjugates or liposomes can be directed, byincorporating the disclosed external domain moiety, to HEVs thatgenerate at the sites of local tumor drainage, to image or disrupt thetumor blood supply.

It is understood that the particular nucleotide and amino acid sequencesdisclosed in FIG. 4 are representative in the sense that counterpart andrelated human genes and alleles can be conveniently and directlyobtained pursuant to this disclosure. For example, cross-hybridizationof the disclosed nucleic acid sequence(s) with genetic material fromhuman cells, particularly lymphocytes present in chronic sites ofinflammation, can be readily performed to obtain equivalent humansequences that hybridize under stringent conditions. In an analogousmanner, degenerate oligonucleotides can be readily synthesized from thedisclosed amino acid sequence, or portions thereof, and amplified usingthe polymerase chain reaction technique to obtain probes that bind toequivalent human sequences. Antibodies directed against the disclosedpolypeptide can also be employed to cross-react with equivalent humanand other mammalian peptides having similar epitope(s).

The following examples are presented to illustrate the advantages of thepresent invention and to assist one of ordinary skill in making andusing the same. These examples are not intended to in any way otherwiselimit the scope of the disclosure or the protection granted by LettersPatent hereon. For experimental details of Examples 1 to 5, see theappended Experimental Procedures section.

EXAMPLE 1 Immunoselection of cDNA clones with antiserum directed againstECMRIII.

RNA from a baboon lymphoid cell line, 594S, which adheres well to HEV,was used to produce a cDNA library in a bacterial expression vector,λSJ349. λSJ349 is based on λgt11 but accepts cDNA fragments in adirectional fashion. cDNA fragments are inserted into the LacZ gene ofthe vector, resulting in the production of β-galactosidase-594S fusionproteins. Exhaustive probing of the library with monoclonal antibodies(P3H9, P1G12, Hutch-1) against the gp90 adhesion receptor wasunsuccessful, indicating that these antibodies may recognizecarbohydrate- or conformation-dependent epitopes. However, a rabbitpolyclonal antiserum, R1594, raised to and then affinity purified withhuman ECMRIII, detected 12 independent antigen-expression phages of theapproximately 1.5×10⁵ clones that were screened. Nucleic acidhybridization data revealed that 8 of the 12 clones contained relatedDNA sequences; these 8 clones were further analyzed. A second screeningof the library was performed using one of these clones, A10.3, as ahybridization probe. An additional 21 clones were identified. Clonesidentified in the initial antibody probing were code-named with an Aprefix, while those obtained by hybridization were given a B prefix.

FIG. 1 is a map that indicates the alignment of 22 of the isolated cDNAclones, and structural features of the protein encoded by a prototype B7cDNA clone. The divisions shown on the top line indicate 100 bpintervals. The prototypic clone B7 is placed at the top of the drawing,and the remaining clones are aligned to B7. The cDNA clones are, fromtop to bottom, B7*, B8, B44, B49, B5c, B6*, A10.3, A1.2, B1*, A11.2,B37, B56, B12, A11.1, B36, B23, B38, B34, A9.1f, A10.1*, B57, and B5d*.One clone, B34, extends approximately 1000 bp beyond the right edge ofthe map. All clones are derived from the λSJ349-594S cDNA library. Sixclones (A series) were isolated by virtue of antibody reactivity. Theoriginal antibody reactive-oligonucleotide hybridizing clone A10.3, usedas a hybridization probe in a screening of the λSJ349-594S cDNA library,allowed the isolation of 21 additional clones. 17 of these clones (Bseries) are shown. 12 of these 22 clones (underlined in the list below)produce R1594 antibody-reactive fusion proteins. The boxes delineate thelocations of the independent 5' and 3' epitopes deduced from thelocation of the endpoints of the antibody-reactive clones and epitopeselection analysis. Epitope selection analysis of a select 5 of these 12(marked with an * in FIG. 1) is shown in FIG. 2.

The hydrophilic character of the polypeptide encoded by the B7 cDNAclone is shown above a block diagram of the coding region. Relativehydrophobicity is above and hydrophilicity is below the horizontal zeroline. These data were calculated with a window of 7 amino acids byGenepro DNA analysis software. Potential N-linked carbohydrate sites areshown as --CHO, the locations of Cys residues as thin vertical lines,and the locations of internal Met residues as thick vertical lines. Thepotential signal peptide and transmembrane regions are shown hatched.

EXAMPLE 2 Multiple gp90 epitopes are encoded by the isolated cDNAclones.

The isolation of several nonoverlapping cDNA clones allowed a directimmunological demonstration that the prototype cDNA encodes the bonafide antiserum-reactive gp90 in 594S cells. This was accomplished by anepitope selection analysis (Weinberger, C., et al., Science 228:740-742, 1985) in which nitrocellulose filters containing fusionproteins from individual clones are incubated with the polyclonalantiserum, followed by elution of the adherent antibodies. Theseantibodies were used to probe a transfer blot of electrophoreticallyseparated cellular proteins. Nonoverlapping cDNA clones, bearing nocommon nucleic acid sequences, can present distinct epitopes and shouldimmunoselect mutually exclusive antibody subsets from the antiserum.Epitope selection analysis was performed by incubating the R1594antiserum with several different cDNA clones (A10.1, B5d, B7, B6, andB1) to produce clone-specific antibody subsets, which were then testedfor gp90 reactivity on a Western blot containing wheat germ agglutinin(WGA)-binding proteins from 594S cells. In the resulting immunoblot,shown in FIG. 2, the first lane was probed with crude R1594 antiserum.As a negative control, epitope selection was carried out with thecloning vector containing an irrelevant insert, and the elutedantibodies were used to probe the second lane; no anti-gp90 activity wasdetected. The remaining lanes were probed with the antibodies selectedby the indicated clones; in each case anti-gp90 activity was present.

These data can be most simply interpreted as evidence for at least twoepitopes, one in the 5' and one in the 3' region of the molecule. Theendpoints of the clones shown in FIG. 1 suggest map locations for theseepitopes, designated by the hatched boxes. This analysis demonstratesthat two (or more) distinct polypeptide epitopes expressed by the cDNAclones are shared with the 90 kD glycoprotein, providing compellingevidence for the identification of these gene sequences by serologiccriteria.

Although the intensity of the bands shown in FIG. 2 suggest that theR1594 antiserum has the major portion of its activity directed againstthe carboxy terminal portion of the protein, the relative stabilities ofthe fusion proteins expressed by the clones used in the epitopeselection experiment are unknown and prohibit simple interpretation ofthe signal strength data.

A less intense, higher molecular weight band is also present in alltracks except that of the vector, and may be a differently modifiedversion of the same core protein as gp90. High molecular weight bands(≧180 kD) have often been seen in 594S and other cells usinggp90-specific antibodies in radioimmunoprecipitation and immunoblotanalyses (Jalkanen, S., et al., 1988, supra; Carter, W. G., and E. A.Wayner, 1988, supra. Our data (not shown) suggest that this highermolecular weight product shows extensive amino acid similarity with the90 kD product, sharing at least the two regions of the proteinidentified above.

EXAMPLE 3 Biochemical confirmation of adhesion receptor cDNA identity.

It was important to demonstrate that the isolated cDNA encodes a gp90which is recognized by an adhesion receptor-specific monoclonal antibodyand not an irrelevant gp90 which also is recognized by R1594 serum.Accordingly, Hermes-1-specific gp90 from the 594S cell line was purifiedand subjected to amino acid sequence analysis. The Hermes-1-specificadhesion receptor was purified from baboon 594S cells, subjected toreducing NaDodSO₄ -PAGE (8%), and stained with Coomassie Blue R. Theseresults are shown in FIG. 3A: partially purified protein obtained byomitting the WGA-sepharose selection step (lane 1); purified proteinfrom 10⁹ cells (lane 2); and prestained molecular weight standards (lane3). No sequence data was obtained on the intact protein, indicating thatthe N-terminus was blocked; an alternate strategy was pursued.

The effect of cyanogen bromide treatment was examined on ¹²⁵ 1-labeledgp90 that was immunoprecipitated with Hermes-1-sepharose from a lysateof surface-labeled 594S cells. The bound proteins were eluted withpropionic acid, treated with cyanogen bromide, and then electrophoresedthrough polyacrylamide. The autoradiogram in FIG. 3B shows the elutedmaterial with (lane +) or without (lane -) subsequent cyanogen bromidetreatment. Cyanogen bromide cleavage generates a 60 kD product. Nosmaller fragment(s) was apparent, presumably because it lacked ¹²⁵I-labeled tyrosines.

Purified, unlabeled protein was then treated with cyanogen bromide toproduce the 60 kD fragment in sufficient quantity for amino acidsequence analysis. Automated sequencing yielded the sequenceMet-Val-Lys-Ala-Leu-Ser-Ile-Gly-Phe. This protein sequence informationwas utilized to synthesize a 256-fold degenerate 27-nucleotide oligomerwhose sequence was chosen based on human codon usage tables and nearestneighbor analysis (Lathe, R., J. Mol. Blol. 183: 1-12, 1985). Theoligomer was end-labeled with ³² p and used to probe the A series cDNAclones that were identified in the initial screening of the library withR1594 antiserum. The probe hybridized under stringent conditions to the5' end of clone A10.3 (data not shown). The nucleic acid sequence ofthis region of A10.3 was found to encode these amino acids of thepurified protein with one exception, Glu instead of Val at position #2(amino acids 66-74 in FIG. 4). These results confirm that this cDNAencodes this adhesion receptor for HEV. Thus the same cDNA clones wereidentified by independent immunological and biochemical criteria.

EXAMPLE 4 DNA sequence analysis of the adhesion receptor cDNA.

The complete DNA sequence of the B7 cDNA clone is shown in FIG. 4. TheDNA sequence and the translated protein sequence of B7 cDNA clone isshown. The numbers at the right margin designate nucleotide and aminoacid numbers. The putative signal peptide (AA1-20) and the putativetransmembrane segment (AA271-290) indicated in FIG. 4 are shown shadedin FIG. 1. Consensus asparagine linked glycosylation sites are boxed.Cysteine residues are bold capitalized. Noncoding nucleotides are shownin lower case.

The B7 cDNA clone was chosen as the prototypic sequence based on itslength and its 5' DNA sequence. The 1537 nucleotide cDNA contains onelong open reading frame of 1086 bp. The 5' and 3' untranslated sequencesare 121 and 330 nucleotides in length, respectively. This candidateprotein coding region begins with the first Met residue encoded in anyof the three reading frames. No stop codon is present upstream of theprobable initiator methionine. This start location agrees well with thetranslation initiation consensus sequence {GCC}GCC(A/G)CCATGG! (Kozak,M., Nucleic Acids Res. 15: 8125-8148, 1987). No poly(A) tail orpolyadenylation signal sequence is present, indicating that the mRNAsequence extends 3' of the cloned cDNA fragment. The DNA sequencecontains no significant repeated sequences. No highly significant matchto this sequence is present in the EMBL (release 17) or Genbank (release57) databases.

EXAMPLE 5 The adhesion receptor is a transmembrane protein.

Analysis of the protein sequence deduced from the B7 cDNA DNA sequenceindicates that the primary translation product consists of 362 aminoacids with a predicted MW of 39.4 kD. After cleavage of the probable 20amino acid leader peptide (see FIGS. 4 and 1), the remaining 342 aminoacid protein has a predicted MW of 37.0 kD, indicating that the 90 kDcell surface form of the protein is highly modified. The proposed signalpeptide cleavage site is indicated by both the (-3,-1) rule and by theweight-matrix methods (von Heijne, G., Nucleic Acids Res. 14: 4683-4690,1986; von Heijne, G., Eur. J. Biochem. 133: 17-21, 1983).

Cyanogen bromide treatment of this gene product will generate a largefragment of 263 amino acids (66-329) corresponding to the 60 kD proteinfragment whose N terminal sequence was determined above. This fragmenthas a calculated polypeptide MW of 28 kD, suggesting thatposttranslational modification contributes approximately 32 kD ofapparent MW to this region of the molecule.

A striking putative transmembrane sequence of approximately 20 aminoacids (see FIGS. 4 and 1) begins after a mature external domain of 250amino acids. The cytoplasmic domain of 72 amino acids is highly charged(32% charged residues). The relatively hydrophilic external domaincontains only 10% charged amino acids. The C terminal 50% of theexternal domain is significantly more hydrophilic than the N terminal50% (FIGS. 1 and 4.

The mature protein contains 7 potential sites of attachment ofasparagine linked carbohydrate distributed along the mature externaldomain of the protein in general agreement with the number (8-10) of Nlinked sugars determined for the human product (Carter, W. G., and E. A.Wayner, 1988, supra).

The mature protein contains 8 Cys residues (FIG. 1, thin lines; and FIG.4, caps). One of these residues is located in the putative transmembranesegment while another is positioned just inside the cytoplasmic domainnear the membrane where it may be available for membrane attachment viaa thioester bond to a fatty acid. This residue, Cys₂₉₆, is flanked onthe N terminal and C terminal sides by 3 Arg and 3 Lys residues,respectively. These charged residues may interact with the negativelycharged phospholipids on the cytoplasmic side of the membrane bilayer.Similar sequences are found in VSV G protein whose Cys residue isacylated with palmitic acid (Rose, J. K., et al., Proc. Natl. Acad. Sci.USA 77: 3884-3888, 1980).

The remaining 6 Cys residues are spaced throughout the N terminal 44% ofthe external domain. At least one pair of these residues probably formsa disulfide bond, as indicated by the decrease in mobility duringelectrophoresis under reducing conditions (Jalkanen, S. T., et al.,1986b, supra). These observations, the presence of multiple Cysresidues, and the electrophoretic mobility behavior suggest that the Nterminal half of the external domain of the molecule may be a relativecompact, folded structure.

The protein lacks an RGD sequence, consistent with our experimentsindicating that lymphocyte adhesion to high endothelium does not involvethe RGD recognition sequence (data not shown). The protein sequence ofthis glycoprotein is unique in the Protein Identification Resource (PIR,release 16) database and both the EMBL and Genbank DNA databasestranslated into all 6 reading frames. An intriguing sequence similarityin the PIR database was found with mouse link protein, a hyaluronic acidbinding protein present as a structural component of proteoglycans(Doege, K., et al., Proc. Natl. Acad. Sci. USA 83: 3761-3765, 1986).This similarity (a match of 28/101 residues, or a match of 41/101including conservative amino acid changes, and 20% exact matchesoverall) includes 4 of the Cys residues of the external domain.

The isolation of the gene sequences encoding the lymphocyte adhesionreceptor/ECMRIII molecule now provides the means for a directdetermination of the structure and function of this abundant cellsurface molecule. Two functions have been determined for this molecule:the adhesion to mucosal HEV which is directly interfered with by mab tothis molecule, and the adhesion of this molecule to Types I and VIcollagen. The structure and role of posttranslational modifications inthe function of this molecule will be facilitated by the manipulation ofthe amino acid sequence. Using molecular genetic methods, one candirectly determine which areas of the molecule participate in adhesionto HEV and to collagen.

The receptor molecule is heavily modified, with the 37 kD corepolypeptide holding 53 kD of apparent MW of posttranslationalmodification. These modifications include both N- and O-linkedglycosylation, addition of the glycosaminoglycan chondroitin sulfateyielding a multitude of larger antigens, addition of sulfate, and theaddition of phosphate to cytoplasmic domain serine residues (Jalkanen,S., et al., 1988, supra; Carter, W. G., and E. A. Wayner, 1988, supra).Other potential modifications may include ubiquitin and a fatty acidaddition. The mouse mab MEL-14 has been shown to recognize thepolypeptide ubiquitin (Siegelman, M., et al., 1986, supra; St. John, T.,et al., 1986, supra). Jalkanen et al. (1987 and 1988, supra) has shownthat this mab also recognizes the human pg90 and gp180-200 products, andthat preclearance with the Hermes-1 antibody removes all MEL-14 reactivematerial. These data suggest that ubiquitin epitope(s) may also resideon the primate molecule, although no evidence for a ubiquitin N-terminuswas found in the protein sequence data derived from either the N-terminus or the largest CNBr fragment. The deduced protein sequencesuggests that the Cys residue located in the cytoplasmic domain near theinner membrane surface may be a point of attachment for a fatty acid.However, the addition of such a molecule would have little effect on theapparent MW of the mature protein.

Although differences in the quantity or quality of these modificationsmay play a direct role in the determination of the organ specificity ofHEV adhesion, variously modified molecules have not been correlated withthe organ specific HEV adhesion phenotype (Jalkanen, M., et al., 1988,supra). Small but important differences may have been overlooked due tothe diffuseness of the 90 kD band. The possibility exists that the moreeasily examined 90 kD molecule is not the actual adhesion structure.Rather, adhesion to mucosal HEV may be facilitated by one of the largerMW forms of the molecule. Modifications of the 180-200 kD forms would goundetected due to the minute effect these alterations would likely havein such a large structure.

In vivo, lymphocyte transmigration from the blood vasculature into theparenchyma of a lymph node is preceded by adhesion to specialized highendothelial cells lining the venules within the node. By definition, theadhesion receptors function in this first stage of adhesion, but howthey contribute to the subsequent extravasation process and whetheradditional lymphocyte surface structures are required for the latterprocess is unknown. In general, cell adhesion and mobility are processesthat involve the cytoskeleton and require interaction with theextracellular matrix (Bretscher, M. S., J. Cell. Biol. 106: 235-237,1988; Damsky, C. H., et al., in The Biology of Glycoproteins, R. J.Ivatt, ed., Plenum Publishing Co., New York, N.Y., p. 1, 1984; Yamada,K. M., Ann. Rev. Biochem. 52: 761-799, 1983). The recent finding thatthe ECMRIII/HEV adhesion receptors, major cell surface proteoglycans,bind the extracellular matrix component, collagen, and are associatedwith the cytoskeleton (Carter, W. G., and E. A. Wayner, 1988, supra)suggests the possibility that they provide a transmembrane link that mayfacilitate adhesion and perhaps even participate in transmigration intothe lymph node. This type of cytoskeletal attachment manifests itself inthe rather low diffusion constant (Jacobson, K., et al., J. Cell. Biol.99: 1624-1633, 1984) exhibited by the cell surface molecule and resultsin its exclusion from coated pits (Bretscher, M. S., et al., Proc. Natl.Acad. Sci. USA 77: 4156-4159, 1980). The approximately 70 cytoplasmicdomain amino acids of the gp90 receptor may be involved in theattachment of this protein to the cytoskeleton.

How does one account for the observations that the proteinsindependently identified as the collagen-binding ECMRIII and theadhesion receptor for HEV are nearly ubiquitous in tissue distributionyet mediate lymphoid organ-specific adhesion of lymphocytes? And whatfunction do these receptors have in other tissue systems? Multiple organspecific adhesion specificities may result from the expression ofindependent receptor molecules whose diversity is controlled by genesequence, gene structure, or from variations in posttranslationaladditions of carbohydrate, ubiquitin, phosphate other moieties, or by acombination of these mechanisms. Equally simple, these organspecificities may be dictated by the sum of the specificities of severalindependent receptor systems (including non-gp90 types). Any of thesemechanisms might be sufficiently cell-type-specific and temporallyregulated to result in the expression of different adhesionspecificities in different cell types or stages of cell maturation. Innonlymphoid cells, the adhesion receptors may be important in cellmobility, tumor metastasis, and embryonic development, phenomena thatinvolve cell adhesion to extracellular matrix proteins (Bretscher, M.S., 1988, supra; Damsky, C. H., et al., 1984, supra; Bretscher, M. S.,et al., 1980, supra; Liotta, L. A., Cancer Res. 46: 1-7, 1986; Thiery,J. P., et al., Ann. Rev. Cell. Biol. 1: 91-113, 1985). Furtherbiochemical characterization of both the lymphocyte and endothelialcomponents involved in recirculation will clarify the molecular basis oftheir interaction and their roles in adhesion and extravasation.

Experimental Procedures

cDNA Library Construction. Poly(A) containing RNA from a baboon lymphoidcell line, 594S (Rabin, H., et al., Intervirology 8: 240-249, 1977), wasreverse transcribed and homopolymer tails added to the 3' end of thecDNA product by terminal deoxynucleotidyltransferase in the presence ofdGTP. A 25 base oligonucleotide, 5'GGGGCGGCCGCCCCCCCCCCCCCCC 3', wasannealed to the tailed cDNA and extended with E. coli DNA polymerase I.This material was ligated with EcoRI adaptors, cleared with NotI, andmaterial larger than≈350 bp purified by gel electrophoresis. This cDNAsynthesis procedure produces cDNA fragments flanked by NotI and EcoRIsites at the 5' and 3' termini, respectively. cDNA was ligated with thepurified left and right arms of λSJ349 DNA that had been cleaved withEcoRI and NotI, in vitro packaged, and amplified by growth on thebacterial strain Y1090.

λSJ349. The λSJ349 vector is a directional cloning version of λgt11produced by the insertion of a 33 bp oligonucleotide(AATTGGGCCCAATGCATTGGCGCCGCGGCCGCG) at the λgt11 EcoRI site. Theorientation of insertion of this oligo as an EcoRI fragment results in asingle NotI site upstream of the single EcoRI site. A 1304 base pairstuffer fragment containing the E. coli rpsL gene allows the EcoRI-NotIdigestions to proceed more efficiently.

Preparation of Rabbit Polyclonal Anti-gp90 Antibodies (R1594). The 90 kDantigen was purified from HT1080 human fibrosarcoma cells as previouslydescribed (Carter, W. G., and E. A. Wayner, J. Biol. Chem. 263:4193-4201, 1988) followed by preparative SDS-PAGE. A New Zealand whiterabbit was immunized with this material. The resulting antiserum wasaffinity purified on immobilized gp90 (purified with the P3H9 mab).

Antibody Screening of Phage Library. The resulting library was platedand the released β-alactosidase fusion proteins transferred tonitrocellulose filters. These filters were incubated with the R1594rabbit serum followed by ¹²⁵ 1-protein A as described previously(Landau, N. R., et al., Proc. Natl. Acad. Sci. USA 81: 5836-5840, 1984).Positive clones were purified by dilution and reprobing.

Epitope Selection and Protein Blotting. Five antiserum-reactiverecombinant clones and the λSJ349 vector (containing an irrelevantinsert) were used for epitope selection essentially as previouslydescribed (Weinberger, C., et al., 1985, supra). Clones were plated,transferred to nitrocellulose filters, and then incubated with crudeR1594 antiserum (diluted 1:100). Filters were washed, and the boundantibodies were eluted in 50 mM glycine, pH 2.5/0.1% NP40/0.15M NaCl,neutralized with 2M Tris, pH 8.0, and used to probe a protein gel blot.WGA-selected 594S cell proteins (prepared as described below) weresubjected to reducing NaDodSO₄ -PAGE, and transferred to anitrocellulose filter. The filter was cut into strips which were probedwith the crude serum or clone-selected antibodies, as indicated. Thestrips were then incubated with ¹²⁵ i-labeled protein A (0.5×10⁶cpm/ml). After washing, the strips were subjected to autoradiography.

Protein Purification and Chemical Cleavage. 594S cells were grown to adensity of 2×10⁶ cells/ml in RPMI1640/10% fetal calf serum and lysed in3% NP40/20 mM Tris, pH 7.4/0.15M NaCl. The lysate was centrifuged at 27krpm in an SW50.1 for 30 minutes at 4° C., and the cleared supernatantwas batch-loaded onto WGA-sepharose. Bound proteins were eluted in 0.2MN-acetylglucosamine in the lysis buffer, and batch-loaded onto sepharoseconjugated with Hermes-1. Proteins were eluted from the immunoaffinitycolumn in 50 mM glycine, pH 2.5/0.3% NP40/0.15M NaCl, then dialyzed andprecipitated with ethanol.

¹²⁵ I-labeled adhesion receptor was immunoprecipitated withHermes-1-sepharose from a lysate of 594S cells that had beensurface-iodinated using lactoperoxidase catalysis. Bound labeledproteins were eluted in 1M propionic acid, then half of the sample wasexposed to cyanogen bromide at ambient temperature overnight. Sampleswere lyophilized in a speed-vac, then electrophoresed through a 12%polyacrylamide gel under reducing conditions and autoradiographed.

Protein Sequence Determination. NaDodSO₄ -PAGE was performed underconditions appropriate for amino acid sequence analysis, proteins weretransferred to derivatized GF/F paper, stained, and the gp90 band wasexcised for automated sequencing as previously described (AppliedBiosystems Protein Sequencer User Bulletin, Issue 25, Nov. 18, 1986).

Oligonucleotide Hybridization. The 27 bp oligonucleotide was end labeledwith ³² p by T₄ polynucleotide kinase. Hybridization was conducted with2.5×10⁶ cpm/ml at 52° C. in 5× SSPE/0.1% Ficoll/0.1% bovine serumalbumen/0.196 polyvinyl pyrrolidone, followed by repeated washing atroom temperature in 5× SSPE. 1× SSPE is 0.18M NaCl/10mM Na₁.5 PO₄ /2mMNa₂ EDTA, pH 7.0.

DNA Sequence Determination. Sequence was determined of both DNA strandsby a combination of ExoIII deletion methods (Henikoff, S., Gene 28:351-359, 1984) and through the use of specific syntheticoligonucleotides located approximately 200 bp apart along the cDNAsequence. Sequencing reactions were performed with Sequenase™ kitreagents (United States Biochemical Corp.) according to themanufacturer's recommendations.

Oligonucleotide Primer Extension. An mRNA complementary oligonucleotide(nuc. 179-208) was synthesized, phosphorylated with λ³² -P-ATP (3000Ci/mmol), annealed to 594S mRNA and extended with reverse transcriptase.380,000 cmp of oligonucleotide and 2 μg of RNA were used. Extensionswere performed with the human cell line Daudi mRNA and the mouse Tlymphoma EL4 mRNA as controls. The length of the visible bands wasdetermined by the electrophoresis of DNA sequencing reactions inadjacent tracks.

EXAMPLE 6 Cell surface expression of the primate adhesion receptor onmouse L cells.

To determine whether B7 encodes an expressible cell surface proteinreactive with the available battery of adhesion receptor-specificantibodies, the B7 cDNA insert was subcloned into a mammalian expressionvector carrying the SV40 early gene region enhancer and promoter, donorand acceptor splice sites, and a polyadenylation signal. Using acalcium-phosphate precipitation technique, this B7 containing plasmidwas cotransfected with a plasmid containing the neomycin resistance geneinto mouse L cells. Sixteen independent stable transfectants expressingthe neomycin resistance gene were selected by growth in mediumcontaining G418. Of these, ten expressed the adhesion receptor on thecell surface as visualized by fluorescence microscopy after stainingwith the anti-ECMRIII monoclonal antibodies, P1G12 and P3H9, as well asthe adhesion receptor-specific Hermes-1 monoclonal antibody. Flowcytometry analysis was performed on a representative L celltransfectant, LB7-6, as well as on nontransfected L cells and the baboonlymphoid 594S cell line. The data demonstrated unimodal staining of thelymphoid cells and the L cell transfectants with each monoclonalantibody, while the nontransfected cells were negative. That p3H9,P1G12, and Hermes-1 all stained the transfectants confirms that eachrecognizes the same B7-encoded surface protein that is expressed in 594Scells. Whether additional gp90 adhesion receptor species are present onthe 594S cell surface is unknown. It is very unlikely that thetransfection procedure itself activates a previously silent mouse geneor alters a normal mouse protein resulting in a detectable product,because not all neomycin-resistant L cell transfectants expressed theadhesion receptor. Nor did cells transfected with the neomycinresistance gene alone stain the adhesion receptor-specific antibodies.

The recognition of the clone B7-encoded protein in L cell transfectantsby the anti-ECMRIII monoclonal antibodies, P1G12 and P3H9, as well as bythe anti-adhesion receptor antibody, Hermes-1, provides geneticconfirmation that these antibodies recognize the same protein. Many ofthe characteristic traits of the ECMRIII/adhesion receptors, includingacidic isoelectric point, size, glycosylation, phosphorylation, andwidespread tissue-distribution, are also exhibited by several other80-95 kD membrane proteins. These include: CD44, and 80 kD proteinassociated with thymocyte maturity, also referred to as p80-A1G3; Pgp-1and related molecules; and a gp85 that is linked to the cytoskeleton viaattachment to ankyrin. It has been suggested that CD44 and theankyrin-binding gp85 are identical to or at least antigenically relatedto Pgp-1. The possibility that these 80-95 kD proteins may be related tothe adhesion receptor for HEV has been raised. To investigate therelationship between these molecules and the clone B7-encoded adhesionreceptor, we examined LBU-6 transfectants, nontransfected L cells, and594S cells for CD44 expression with the anti-CD44 monoclonal antibody,A1G3. The resulting flow cytometry analysis showed that A1G3 stains 594Scells, the transfectant LB7 cell line, but not nontransfected L cells.Thus, the adhesion receptor encoded by clone B7 is a CD44 molecule. Ithas been reported that CD44 is distributed throughout the body, but inthe thymus is restricted to medullary thymocytes where it is acquiredduring T cell maturation. These findings are consistent with the knowndistribution of adhesion receptors.

Biochemical information about the expression of the primate adhesionreceptor in mouse L cell transfectants was obtained byradioimmunoprecipitation analysis on the three most brightly stainingtransformants, LB7-2, LB7-6, and LB7-7, in parallel with 594S cells andnontransfected L cells. Cells were surface iodinated with ¹²⁵ I,solubilized in NP-40 and the lysate was incubated with the P3H9 antibodyand Protein A-sepharose. The bound proteins were subjected to reducingSDS-PAGE and autoradiographed. As before, the 594S protein migrated as adiffuse band around 92 kD. Each of the L cell transfectants produced aP3H9-specific surface protein of 88 kD, indicating a high degree ofposttranslational modification. Differences in glycosylation or othermodifications might account for the small discrepancy observed inprotein size between the mouse transfectants and the baboon 594S cells.

Expression of the primate adhesion receptor for HEV on the surface ofmouse L cells provided the opportunity to determine whether it conferreda new behavior on L cells, the ability to adhere to primate HEV.Adhesion to peripheral lymph node HEV was examined in vitro withB7-transfected L cell lines, nontransfected L cells, and baboon lymphoid594S cells. The test cells, along with directly fluoresceinated 594Scells (as an internal control) were incubated on frozen sections ofprimate peripheral lymph node, washed, fixed, and the adherent cellscounted. The number of adherent test cells was normalized to the numberof adherent internal control cells, providing an adhesion ratio. Theadhesion ratio of the LB7-6 transfectants was 1.5-fold greater than thatof nontransfected L cells, while 594S cells adhered 3.5-fold better.However, the nontransfected L cells themselves exhibited a higher levelof adhesion to the lymph node HEV than is typically observed withnonbinding lymphoid tumors. Perhaps mouse L cells (fibroblasts) expressother adhesion molecules that mediate a basal level of adhesion to HEV,such as a member of the integrin family of adhesion receptors. Thatmouse L cell transfectants do not exhibit a level of adhesion comparableto 594S cells might result from a variety of factors, such as speciesdifferences in the structure and function of accessory molecules, thelack of necessary accessory molecules, and gross morphologicaldifferences in L cell fibroblasts compared with lymphocytes. We arecurrently investigating whether expression of clone B7 in ahematopoietic cell background will confer a level of adhesion comparableto peripheral node-binding lymphoid cells.

EXAMPLE 7

As discussed above, we have identified extensive structural homologybetween one type of heterotypic adhesion receptor (HAR) involved inlymphocyte interactions with high endothellum in lymphold organs and acollagen-binding protein, termed extracellular matrix receptor III(ECMRIII) expressed on most nucleated cell types. Both receptors havebeen described as heterogeneous 90 kDa transmembrane glycoproteins,referred to here as GP90. In the additional experiments summarizedbelow, monoclonal anti-HAR antibodies, Hermes-1 and Hutch-1, andmonoclonal anti-ECMRIII antibodies, P1G12 and P3H9, were utilized tocompare the two receptors. The following observations were made: (i) Allthese monoclonal antibodies (mabs) immunoprecipitated major GP90components as well as uncharacterized additional higher molecular massantigens of 120-200 kDa in human and macaque fibroblast and peripheralblood mononuclear cells (PBMC). (ii) Competitive binding analyses withthe antibodies identified distinct epitopes present on GP90. (iii)Enzymatic and chemical digestions generated identical peptide fragmentsfrom all the antigens in human and macaque fibroblasts and PBMC. (iv)Sequential immunoprecipitation with P1G12 followed by the other mabsindicated that all GP90 species reactive with Hermes-1 and Hutch-1 alsoexpressed the P1G12 defined epitope. In reciprocal experiments, Hermes-1and Hutch-1 immunoprecipitation did not completely remove allP1G12-reactive GP90 from cellular extracts. One inference from thesedata would be that GP90 is serologically heterogeneous encompassing HARSas a major subset of this broadly expressed class of molecules.

While the present invention has been described in conjunction with apreferred embodiment, one or ordinary skill, after reading the foregoingspecification, will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definition contained in theappended claims and equivalents thereof.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. An isolated polypeptidecomprising a fragment of a lymphocyte adhesion receptor selected fromthe group consisting of:(a) amino acid residues 21 through 270 of thesequence set forth in FIG. 4, (b) amino acid residues 271 through 290 ofthe sequence set forth in FIG. 4, (c) amino acid residues 21 through 290of the sequence set forth in FIG. 4, (d) amino acid residues 271 through362 of the sequence set forth in FIG. 4, and (e) amino acid residues 291through 362 of the sequence set forth in FIG. 4,said polypeptide beingfree of proteins from the same mammal.
 2. An isolated polynucleotidesequence encoding the polypeptide of claim
 1. 3. The polynucleotidesequence of claim 2 which is a DNA.
 4. The DNA of claim 3 selected fromthe group consisting of:(a) nucleotides 182 through 931 of the sequenceset forth in FIG. 4, (b) nucleotides 932 through 991 of the sequence setforth in FIG. 4, (c) nucleotides 182 through 991 of the sequence setforth in FIG. 4, (d) nucleotides 932 through 1207 of the sequence setforth in FIG. 4, and (e) nucleotides 992 through 1207 of the sequenceset forth in FIG. 4.